Methods of impregnating tissue samples with solid paraffin are known, are used in industry and, in particular, also in medicine in order to embed tissue samples, after their fixation, dewatering and soaking with paraffin, in paraffin blocks and to prepare these in this manner for a subsequent microscopic examination (histology). The thinnest sections or slices can then be obtained from the tissue samples embedded in paraffin blocks which sections can be examined under a microscope. The term "tissue samples" is to be understood in the sense of the invention as tissue samples in general, namely, human, animal or plant tissue samples.
The tissue samples in a known method (German OS No. 30 47 417), which tissue samples are fixed with a suitable fixing medium (for example, isopropyl alcohol) and are subsequently dewatered through isopropyl alcohol (isopropanol), are impregnated with paraffin without using an intermediate medium (for example xylol), and are thereafter also embedded in paraffin. The impregnating in paraffin takes place at ambient pressure and at a temperature slightly higher than the melting point of the paraffin. In the case of the usually utilized paraffins, the melting point lies approximately between 50.degree. C. and 58.degree. C. To soak the tissue samples, they are dipped into or under the liquid paraffin, namely, are surrounded by the liquid paraffin. The replacement of the isopropyl alcohol contained in the tissue of the tissue samples with paraffin is effected by dissolving the isopropyl alcohol in paraffin such that, during the course of the treating or impregnating time, the concentration of the paraffin in the "isopropyl alcohol-paraffin solution" increases also in the tissue of the tissue samples. With this known method it is not possible, or is possible only over a very long impregnating time, to completely replace the isopropyl alcohol in the tissue of the tissue samples with paraffin or to replace it with paraffin to the degree necessary to achieve adequate quality of the embedded paraffin and thus also necessary for the quality of the tissue sections.
In addition to long impregnating times, the known methods have, among others, also the disadvantage that a considerable contamination of the paraffin with isopropyl alcohol occurs, which means either that additional expensive measures are required for cleaning the paraffin or, on the other hand (because of the necessary paraffin replacement), an increased consumption of paraffin.
The purpose of the invention is to provide a method for impregnating or soaking tissue samples with paraffin, which method avoids the aformentioned disadvantages and, among other advantages, enables a quick and economical impregnating and also embedding of tissue samples in paraffin.
To attain this purpose, a method according to the invention includes the soaking of the tissue sample in a closed, evacuated working chamber under the influence of ultrasound (ultrasonic vibrations) on the tissue sample and on the liquid paraffin surrounding the tissue sample. A vacuum is provided in the working chamber such that under the pressure existing in the working chamber, the boiling point of the isopropyl alcohol is equal to or is below the operating temperature in the working chamber. The operating temperature suitably lies in the range between about 52.degree. C. and about 60.degree. C.
The method of the invention has, amongst others, the advantage, compared with known methods, that significantly shorter impregnating times can be achieved, that is, impregnating times on the order of magnitude of 20 to 30 minutes, for tissue samples of lesser thickness, so that "high-speed paraffin sections" can be obtained utilizing the method of the invention, which sections, for example, during an on-going surgery permit a microscopic examination and diagnosis of a tissue sample and thus, due to this examination, enables suitable surgical steps to be taken during the surgery. The method of the invention has furthermore the advantage that contamination of the paraffin with isopropyl alcohol and thus replacement of paraffin are minimized or avoided, so that the inventive method is also very economical.
The method of the invention achieves also a reduction of the operating temperature, below the melting temperature of paraffin at atmospheric pressure, by applying a vacuum to the working chamber. Thus, it is possible to soak tissue samples with paraffin at very low temperatures, so that also temperature-volatile substances are maintained in the tissue samples and can be identified/traced during subsequent examinations. Their identification is not possible in the known methods.
Aside from a quick, optimum soaking, the method of the invention opens up also new possibilities in the diagnostics field. For example, the method of the invention maintains the operating temperature, the ultrasound treatment and the reduced pressure in the working chamber, which pressure lies, for example, in the order of magnitude of 100 mbar, constant over the entire impregnating time.
The use of isopropyl alcohol also has the particular advantage that whereas this alcohol does escape from the respective tissue sample in the evacuated working area through boiling, said escape takes place so slowly that the paraffin has enough time to penetrate into the tissue sample, and in this way an optimal impregnation of the tissue sample with paraffin is achieved, in particular even in tissue samples--e.g. fat tissues--where impregnation with paraffin calls for a particularly careful mode of operation. The optimal impregnation is further supported in such a way that during impregnation or during the period of treatment of the tissue sample the pressure is increased at least once above the reduced pressure, and then again decreased to the reduced pressure. In so doing the pressure is preferably increased to atmospheric pressure. The total time during which an increased pressure is present is preferably 30-40% of the duration of treatment. During the change in pressure the action of ultrasound on the tissue is preferably maintained.